![]() The PD structure lacks a potential endonuclease domain. However, in a further paper, an alternative explanation of the results was provided which made the authors doubt the existence of trimming endonuclease activity. Initially, it was suggested that Pol θ performs end trimming using the intrinsic endonuclease activity associated with the PD (repurposing of metal ions in the polymerase active site for endonucleolytic cleavage). Nonhomologous regions should be removed and the 3′-tails should be shortened before initiation of DNA synthesis from annealed microhomologies. ![]() The presence of nonhomologous segments flanking the microhomologies at the 3′-ends complicates TMEJ. The alternative clamp RAD9-RAD1-HUS1 complex (9-1-1), Fanconi anaemia group D2 protein (FANCD2) and 5-hydroxymethylcytosine-binding protein (NMCES) possibly also play a role in the recruitment of Pol θ to DNA ]. It was suggested that PARylation can promote the dissociation of Pol θ from DNA strands and termination of DNA synthesis in the end of TMEJ. However, PARP1-dependent PARylation of the Pol θ HD in vitro decreases the ssDNA-binding affinity of Pol θ. Chromosomal TMEJ assays revealed that PARP1/PARP2 deficiency reduces TMEJ activity only 2–4-fold through a decrease in the 5′-3′ resection of DSB, and, thus, re-channels repair to NHEJ. Competing with Ku proteins for DNA ends binding, PARP1 promotes a-EJ, and its inhibition impairs Pol θ recruitment to DNA break sites. PARP1 is activated upon binding DNA strand breaks and performs post-translational modification (PARylation) of many DNA repair proteins. TMEJ is also regulated by poly(ADP) ribose polymerase 1 (PARP1). melanogaster revealed that Pol θ is a critical factor of Lig4 (DNA ligase 4)-independent alternative end joining (a-EJ). Goff et al., demonstrated the sensitivity of POLQ-defective bone-marrow cells to γ-radiation and bleomycin, suggesting the role of Pol θ in DSB repair. About 90% of chaos1 homozygous mice with an ATM-deficient background died during the neonatal period and surviving animals exhibited severe growth retardation and chromosome instability which was synergistic to the ATM-deficiency phenotype. The analysis of Polq chaos 1/ Polq chaos 1 mice with an ATM (ataxia telangiectasia mutated) deficiency allowed the suggestion that Pol θ might be involved in an alternative DSB repair pathway distinctive from the major HR ATM-dependent pathway. The phenotype-based screening of mutant mice showed that the missense mutant allele chaos1 (a C>T substitution at nucleotide 5794 in the coding region of exon 19) of the Polq gene leads to a spontaneous and radioactivity-induced increase in micronuclei number in erythrocytes. It took more than a decade to uncover the molecular basis underlying the role of Pol θ in DSB repair. Īt present, the importance of Pol θ for DSB repair is well-established. The inactive exonuclease-like subdomain, lacking catalytic residues essential for metal-ion binding and primer degradation, has two conserved loops. These contacts can facilitate DNA lesions bypass and the extension of poorly annealed DNA termini during TMEJ. The thumb residues K2181 and R2202 as well as the Insert 2 R2254 residue make unique upstream contacts with the 3′-terminal primer phosphates (n-1–n-5). These elements play a crucial role in the TLS activity and the ability of Pol θ to use primers with only minimal homology to DNA templates during TMEJ. Insert 2 and Insert 3 are located in the palm subdomain and are comprised of 58 and 33 amino acids, respectively. Its proximity to DNA suggests a role in DNA binding. Insert 1 consists of 31 amino acid residues between the first and second conserved motifs and is located in the thumb subdomain. These insertion loops are not found in the two other A-family polymerases. Three conserved loop elements are located within the PD. Importantly, the HD forms a tetramer (a dimer of dimers) both in the crystals and in solution, providing the structural basis for the bridging and annealing of DNA strands by Pol θ. Purified HD is able to unwind different types of DNA with 3′-5′ polarity (in an ATP-dependent manner) and reanneal them. DNA-dependent ATPase activity in vitro is associated with the HD. melanogaster, Caenorhabditis elegans, and even archaea. The HD sequence similarity is observed among Homo sapiens, Mus musculus, D. Among them are the nucleotide-binding site and two RecA-like subdomains (residues 1–289 and 290–513) containing motifs I–VI, which are conserved across SF2 helicases. The HD is composed of five structural elements. The high-resolution cryo-EM structure of the PD of Lates calcarifer Pol θ is also available. Crystal structures of the separate SF2 HD and polymerase domain (PD) of human Pol θ have been solved. Unlike other DNA polymerases, Pol θ contains a separate superfamily 2 (SF2) helicase-like domain (HD) located on the N-terminus of the protein.
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